471-479 pascual.qxd

Acta Neurobiol Exp 2007, 67: 471479
Effects of neonatal maternal deprivation
and postweaning environmental complexity
on dendritic morphology of prefrontal
pyramidal neurons in the rat
Rodrigo Pascual1 and S. Pilar Zamora-León2
1Laboratory of Neuroscience, School of Kinesiology, Faculty of Basic Sciences
and Mathematics, Catholic University of Valparaiso, Avenida Brasil 2950,
Valparaíso, Chile; 2Laboratory of Developmental Neurobiology, Institute of
Basic Science, Catholic University of Maule
Abstract. It has been reported that periodic maternal separation in rats leads to
a variety of endure behavioral, neurochemical and microstructural sequelae
associated with the pathophysiology of anxiety disorders. Since it has been
proposed that these changes might be permanent, we examined whether
environmental complexity aid to recover the structural dendritic impairment
induced by neonatal maternal deprivation in the medial prefrontal cortex of the
rat. In addition, the anxiety-like behavior was assessed in the elevated plus-
maze. Repeated maternal separation between postnatal days 621 (3 hours
daily) significantly reduced the dendritic material in layer II/III pyramidal
neurons and induced anxiety-like behaviors in the elevated plus maze.
Furthermore, environmental stimulation (twice a day, 1 h each) during
12 consecutive days (postnatal days 2335) failed to recover the neuronal and
behavioral disorders induced by neonatal maternal separation. The results
demonstrated that (i) neonatal maternal separation severely altered pyramidal
dendritic outgrowth in close association with anxiety-like behavior assessed in
the elevated plus maze, and (ii) postweaning environmental complexity was
unable to recover neither the prefrontocortical neuronal impairment nor the
novelty-induced anxiety-like behavior triggered by early maternal deprivation.
Correspondence should be
addressed to R. Pascual,
Key words: dendritic impairment, environmental complexity, maternal
separation, medial prefrontal cortex, elevated-plus maze
472 R. Pascual and S.P. Zamora-León
tion may compensate neuronal impairments induced
by neonatal MS. Thus, the objective of the present
Clinical studies have suggested that disruption of the study was to determine (i) whether MS alters dendritic
mother-infant socioemotional bond during the first length and branching of mPFC pyramidal cells, which
year of life constitutes a severe early life stressful are involved in the regulation of complex socio-emo-
experience that may contribute to the pathophysiology tional behaviors, and (ii) if postweaning EC amelio-
of some psychiatric disorders, such as anxiety sub- rates the MS-induced neuronal impairments. In addi-
stance abuse disorders and antisocial behavior (Foley tion, we evaluated the effect of both MS and EC on the
et al. 2004, Lipman et al. 2001, MacMillan et al. 2001). anxiety-like behavior in the elevated plus-maze, the
In addition, several studies performed with laboratory most widely screening assay employed for anxiolytic
animals have clearly demonstrated that repeated mater- and anxiogenic agents in rodents.
nal separation (36 h) during the first 23 weeks of life
have long-term consequences on endocrine, behav-
ioral, and brain development later in life. For example,
maternal separation (MS) in rats showed stress hyper-
Animals and experimental design
reactivity, anxious behavior in the elevated plus-maze
test (EPM), anhedonia, increased ethanol consump-
Adult female Sprague-Dawley rats were mated with
tion, and hyper-reactivity of the hypothalamic-pitu- colony breeder male rats. One week before birth, dams
itary-adrenal (HPA) axis in response to stress (Caldji et were individually housed in standard laboratory
al. 2000, Francis and Meaney 1999, Hofer 1996, Huot Plexiglas cages (40 × 30 × 18 cm), with sawdust as
et al. 2001, Wigger and Neumann 1999). Most of these bedding in an air-conditioned room under standard
behavioral and endocrinal dysfunctions persist laboratory conditions: 12 h light-dark cycle (lights on
throughout adulthood (Daniels et al. 2004). In addtion, at 7:00 AM), 22 ± 2°C, and free access to pellets and
it has been shown that these long-term effects induced water. The date of birth was designated as postnatal
by MS appear to depend upon changes in the structure day 0 (P0). One day after delivery, litters (1014 pups)
and function of the medial prefrontal cortex (mPFC) were cross-fostered and culled to 10 pups each
neurons, which are involved in the regulation of the (5 males and 5 females) and housed together with
stress response (Spencer et al. 2005, Williams et al. a mother in standard rat cages (50 × 30 × 20 cm). Half
2006). In fact, mPFC pyramidal neurons of MS rats of the litters were randomly assigned to the maternal
showed changes in dendritic branching, spine density, separation group (MS, n=20) and the other half to the
and monoaminergic fiber innervation (Bock et al. mother-reared control group (MR, n=20). MS pups
2005, Helmeke et al. 2001, Ovtscharoff and Braun were separated every day from their dams for 3 h
2001, Poeggel et al. 2003), indicating that early (1:004:00 PM) from P6 until P21. During that time
adverse socioemotional experiences, such as maternal MS pups were kept individually in small aluminium
deprivation, interfere with the development of neu- foil nests (12 cm diameter, 15 cm height) filled with
ronal and synaptic composition of the mPFC.
clean bedding and placed on a 36°C heating blanket.
On the other hand, several reports in laboratory ani- There was no visual, tactile, olfactory or auditory
mals indicated that environmental complexity (EC) communication between the pups during the depriva-
enhanced the individuals brain and behavioral devel- tion period. After isolation, the pups were placed back
opment. For instance, rats exposed to EC showed an in their home cage. MR pups were left undisturbed,
increase in brain weight, cortical depth (Rosenzweig except for cage cleaning 2 times a weak. At P21, all
and Bennett 1996), dendritic outgrowth (Green et al. animals were weaned and housed in groups of 4 rats
1983), synaptogenesis (Comery et al. 1996) and neuro- per cage under standard laboratory conditions. At P23,
genesis (Olson et al. 2006). Additionally, it has been half of the MS (n=10) and MR (n=10) animals were
demonstrated that environmental stimulation during randomly selected (MS-EC and MR-EC groups) and
the postweaning period reverses the effects of MS on transferred to an enriched environment (EC), twice
both HPA axis and behavioral responses to stress a day for 1 h each during 12 consecutive days
(Francis et al. 2002). However, there are no studies (P23P35). The EC consist of a large Plexiglas/alu-
examining whether a similar environmental manipula- minum cage (100 × 100 × 70 cm) with a variety of

Neonatal maternal deprivation 473
Fig. 1. Experimental design. (P1P35) postnatal days; (MR)
maternally-reared animals; (MS) maternally-separated ani-
mals; (MR-EC) maternally-reared animals submitted to
environmental complexity; (MS-EC) maternally-separated
animals submitted to environmental complexity; (EPM) ele-
vated-plus maze.
objects, such as tunnels, shelves, running wheels, lad-
ders, and different kinds of manipulable objects (e.g., Fig. 2. Representative Golgi-impregnated pyramidal neuron
jars, glass balls, wooden objects) that where changed of the rats mPFC. Scale bar is 45 mm.
twice a week in order to avoid habituation. At P36, all
animals were behaviorally evaluated and, at the fol-
Neuronal evaluations
lowing day, sacrificed under deep pentobarbital anes-
thesia (50 mg/kg, i.p.). The experimental design is
In order to study the impact of periodic MS on
shown in Fig. 1.
mPFC pyramidal dendritic development, the brains
All experimental protocols followed guidelines were stained with the Golgi-Cox-Sholl procedure
given at Principles of laboratory animal care (NIH (MR, n=10; MS, n=10; MR-EC, n=10; MS-EC, n=10;
publication No. 86-23, revised 1996) and were Sholl 1953; Fig. 2), technique that stains 15% of ran-
approved by the Institutional Animal Care and Use domly distributed neurons (Pasternak and Woolsey
Committee at Universidad Católica del Maule.
1975). After 60 days of slow impregnation, brains were
dehydrated in ethanol-acetone and ethanol-ether solu-
Elevated plus maze
tions (50%/50%), embedded in celoidin, and hardened
in chloroform vapors. Coronal sections (120 µm thick-
At P36, all animals were evaluated in the elevated ness) were cut using a sledge microtome and mounted
plus maze (EPM). The EPM was made of black with DPX (Fluka).
Plexiglas and consisted of 2 open arms (50 × 15 cm)
In order to qualify for the morphometrical evalua-
and 2 closed arms (50 × 15 × 20 cm). The apparatus tions, pyramidal cells should fulfill the following crite-
was mounted on a fixed base, 50 cm above the floor. ria: (i) have a well-defined pyramidal shape, (ii) show
Each rat was placed in the center of the EPM facing an an adequate staining of the soma and dendrites,
open arm, and was allowed to freely explore the EPM (iii) have no extensive processes overlapping neigh-
for 5 minutes. The number of entries and the time boring neurons, and (iv) be located in a cortical strip
spent in the open arms was recorded. Measures were between 100 and 350 mm under the pial surface
carried out under dim light condition between (2.2 mm and 3.2 mm anterior to bregma; Paxinos and
8:0011:00 PM by 2 observers, who were blind to Watson 1998). The first 1012 cells per brain section
the rearing condition of the pups. The EPM was meeting the above criteria were traced with the aid of
cleaned between each pup with 5% ethanol to remove a camera lucida (Olympus, model BH-DA-LB, 400×
odor cues. This test has been validated in rodents to magnification). Two dendritic variables were quanti-
evaluate anxiety related behaviors (Andreatini and fied: (i) the mean basilar dendritic length/neuron by
Bacellar 2000).
means of an electronic map reader (Pascual et al.

474 R. Pascual and S.P. Zamora-León
Fig. 4. Effect of maternal separation and environmental
complexity on basal dendritic length. (MR), (MS) maternal-
ly-reared and maternally-separated animals, respectively;
(MR-EC), (MS-EC) maternally-reared and maternally-sepa-
rated animals submitted to environmental complexity,
respectively. *P<0.05, **P<0.01 (means ± SD).
Fig. 3. Effect of maternal separation and environmental
complexity on (A) time spent (s) and (B) number of entries
into the open arms of the elevated-plus maze. (MR), (MS)
maternally-reared and maternally-separated animals, respec-
tively; (MR-EC), (MS-EC) maternally-reared and maternal-
ly-separated animals submitted to environmental complexi-
ty, respectively. **P<0.01 (means ± SD).
Fig. 5. Effect of maternal separation and environmental
1996), and (ii) the mean basilar dendritic ramifica- complexity on basal dendritic branching. (MR), (MS) mater-
tion/order, determined according to the method of nally-reared and maternally-separated animals, respectively;
Coleman and Riesen (1968). This method states that (MR-EC), (MS-EC) maternally-reared and maternally-sepa-
dendrites coming out directly from the cell body are rated, animals submitted to environmental complexity,
considered as first order, direct branches from first respectively. *P<0.05, **P<0.01.
order dendrites are considered as second order, and so
on. A total of 391 cells were sampled from mPFC (MR: time and did not enter the open arms as many times as
87; MS: 98; MR-EC: 94; MS-EC: 112). All cells were the MR control rats (Fig. 3A and B, respectively;
drawn and analyzed by the same person in a blind man- P<0.01). Unexpectedly, these behavioral alterations
ner to maximize reliability.
appeared to be permanent, since MS environmentally
stimulated animals (MS-EC group) did not differ from
Statistical analysis
MS non-stimulated rats (Fig. 3A and B). On the other
hand, MS rats displayed a decrease in dendritic length
Unpaired two-tailed t-test statistical analyses were compared to the MR control group ( 28%; P<0.05;
performed. Results were expressed as means ± SD and Fig. 4). Again, the EC condition was unable to amelio-
differences were considered significant when P<0.05.
rate the dendritic impairment induced by MS ( 44%;
P<0.01; Fig. 4). Surprisingly, MS did not affect the
number of dendritic branches per neuron (Fig. 5). Only
control rat pyramidal cells submitted to EC condition
Rats exposed to maternal separation (MS group) did (MR-EC group) showed a significant increase in den-
not explore the open arms of the EPM as much as the dritic branching (dendritic orders 47; P<0.05,
control rats (MR group). In addition, MS rats spent less P<0.01; Fig. 5). Those results indicate that post-

Bad Axe, Michigan Wednesday, December 28, 2011 The regular meeting of the Huron County Board of Commissioners was held on Wednesday, December 28, 2011, commencing at 10:15 a.m. in the Board of Commissioners room, Third Floor, Huron County Building, Bad Axe, Michigan. PRAYER AND PLEDGE: The meeting was called to order by Chairman Wruble with The Lord's Prayer and Pledge to the Flag of the United States of America. ROLL CALL: Commissioners present: Chairman Ron Wruble, Steve Vaughan, John Nugent, Clark Elftman, John Horny, John Bodis and Dave Peruski. AGENDA: Commissioner Elftman moves Consent Resolution #31 to the New Business agenda. Motion by Vaughan, seconded by Bodis to approve the agenda as amended. Motion carried. APPROVAL OF MINUTES: Motion by Elftman, seconded by Bodis to approve the Committee of the Whole minutes of December 13, 2011 and the minutes of the regular meeting of December 13, 2011 and Committee of the Whole minutes of December 20, 2011. Motion carried. COMMUNICATIONS: Chairman Wruble turns the following communications over to proper committee.